Studies in rodents show that the gut microbiome influences neurodevelopment and subsequent anxiety-related behaviors which are relevant to a wide range of psychiatric illnesses. However, there is a fundamental gap in translating animal data into the clinic: no study has directly tested whether differences in microbial colonization impact anxiety-related behavior in humans. Furthermore, the mechanisms and pathways by which microbiota alter brain development are poorly understood. Our long-term goal is to determine how colonization of the gut microbiome impacts human brain development and later risk for psychiatric illness. The objective of this application is to determine how microbial colonization impacts anxious behavior at 1 year of age and to identify signaling mechanisms and neural circuits mediating this relationship using high resolution magnetic resonance imaging (MRI), diffusion tensor imaging (DTI) and resting state fMRI (rfcMRI). The rationale for the proposed research is that modulation of the gut microbiota could normalize neurodevelopmental trajectories early in the disease process, ultimately preventing the onset of psychiatric illness o reducing its severity. We will achieve our objective through 5 specific aims. In the R21 phase we will: 1) Confirm that sufficient bacterial diversity is present in fecal samples at 2 weeks and 1 year of age to test relationships with anxious behavior, brain development, and hypothesized signaling mechanisms; and 2) Confirm that hypothesized signaling mechanisms can be successfully probed at 2 weeks and 1 year of age. In the R33 phase we will: 3) Determine how patterns of microbial colonization in infancy relate to anxious behavior at 1 year of age; 4) Identify neural circuits which mediate associations between gut microbiota and anxious behavior in human infants; and 5) Determine the signaling mechanisms by which microbiota affect neurodevelopment and anxious behavior in human infants. Our central hypothesis is that anxiety-related behaviors will differ between infants with different patterns of bacterial colonization and this relationship will be mediated by changes in the amygdala, hippocampus, and medial prefrontal cortex. We further hypothesize that microbiota will impact neurodevelopment by altering pro-inflammatory cytokines and cortisol reactivity, potentially through synergistic effects on the kynurenine arm of the tryptophan metabolic pathway. The application is innovative in that it will be the first study to test if and how microbial compositin relates to anxious behavior in a human cohort. The proposed research is significant in that it is an essential first-step in developing novel interventions to promote a healthy microbiome and reduce risk for psychiatric illness.